Name: measuring post-stroke cerebral edema, infarct zone and blood-brain barrier breakdown in a single set of rodent brain samples
Uploaded: 2020-10-23T13:30:00.000+00:00
Duration: 4 min 32 s
Description: Watch this Scientific Journal Video about Measuring Post-Stroke Cerebral Edema, Infarct Zone and Blood-Brain Barrier Breakdown in a Single Set of Rodent Brain Samples at JoVE.com

我们感谢马里娜·库舍里亚瓦、马克西姆·克里沃诺索夫、达里娜·亚库门科和叶夫根尼亚·贡查里克，他们支持我们的讨论，并为我们的讨论作出了有益的贡献。获得的数据是鲁斯兰·库茨博士论文的一部分。

下列程序是根据《赫尔辛基宣言》和《欧洲共同体实验动物使用准则》的建议进行的。内盖夫本古里安大学的动物护理委员会也批准了这些实验。
1. 为实验程序准备大鼠
<ol><li>选择成年雄性斯普拉格-道利大鼠，没有明示病理学，每只体重在300至350克之间。</li>
	<li>将所有大鼠在室温下保持在22°C，在实验前有12小时的光和暗周期。</li>
	<li>确保食物和水是可用的广告利比妥姆。</li>
	<li>执行下午 6:00 至下午 2:00 之间的所有程序.m.m。</li>
</ol>2. 准备大鼠做手术
<ol><li>用异氟化麻醉大鼠30分钟（感应4%，保养2%）和24%氧气（1.5升/分钟）。<ol>
<li>通过确保大鼠没有踏板取出反射来测试大鼠的麻醉水平。</li>
	</ol>
</li>
	<li>将 24 度导管插入尾静脉。 注:不执行血管化的尾部加热。<ol>
<li>把老鼠放在桌子上的姿势。用医用胶带把老鼠的四肢都贴上。</li>
	</ol>
</li>
	<li>手术前将探针放入大鼠直肠进行温度测量。</li>
	<li>在过程中，保持加热板以支持 37 °C 的核心体温。</li>
	<li>在老鼠的眼睛里加入软膏以保护。</li>
	<li>剃光手术区，消毒三种应用10%的波维酮碘，其次是70%的异丙醇。</li>
</ol>3. 右侧中脑动脉闭塞
注:MCAO采用修改后的技术，如先前描述的16、22、23，使用麦加里等人描述的仪器24和乌卢埃等人25。
<ol><li>用手术钳子解剖颈部腹中线的皮肤和表面筋膜，用弯曲的刀片剪刀。</li>
	<li>识别肌肉三角形，由ICA、外部胡萝卜动脉（ECA）和普通胡萝卜动脉（CCA）组成。</li>
	<li>小心地将右CCA和ICA与迷走神经与血管手术的微力分离。</li>
	<li>揭露正确的 CCA 和 ICA 。使用微型夹子或血管手术的特殊环状物阻止从CCA到ICA的血流。使用显微剪刀在ICA上切口（约1毫米），用于血管手术。</li>
	<li>直接通过ICA插入单丝导管（4-0尼龙），从右CCA的分叉点约18.5-19毫米进入威利斯圆，直到达到轻微的阻力，以遮挡MCA26。</li>
	<li>在ICA的分裂之上，在ICA周围展开。</li>
	<li>对于假操作的对照组，执行尼龙线的插入，而不是步骤3.5和3.616，22。</li>
	<li>通过腹膜注射管理5 mL的0.9%氯化钠。</li>
	<li>用缝合关闭伤口，将老鼠带到恢复区。 注意:麻醉结束后几分钟，老鼠会醒来，在笼子里独立移动。</li>
	<li>MCAO后23小时，通过针管27将2%的埃文斯蓝色盐水（4 mL/kg）23，26注入两个手术组的尾静脉。 注:这被用作血脑渗透度示踪器。允许循环60分钟。</li>
</ol>4. 确定梗塞区
<ol><li>测量IZ在24小时后，MCAO如前所述9，15，18，19，26。 注:体重减轻超过20%或出现癫痫发作或六肢瘫痪的老鼠被排除在实验之外。</li>
	<li>用20%的氧气和80%的二氧化碳代替受启发的气体混合物，直到大鼠停止自发呼吸，从而对大鼠实施安乐死。</li>
	<li>用剪刀和手术钳子在肋骨笼下的腹部壁上用5-6厘米的横向切口打开胸部。</li>
	<li>用剪刀和手术钳沿着肋骨笼的整个长度进行隔膜切口。</li>
	<li>小心地取代肺部，切开肋骨笼，直至左右两侧的锁骨28。</li>
	<li>通过心脏左心室注入200 mL的正常盐水。</li>
	<li>用剪刀刺穿或切入心脏右中庭。</li>
	<li>使用断头台进行斩首，并收集脑组织。</li>
	<li>使用虹膜剪刀，从前兆切割到两侧后头骨表面的分离边缘。</li>
	<li>将嗅灯泡、腹腔表面的神经连接和头骨的鼻孔表面与大脑分开。</li>
	<li>从头部取出大脑。</li>
	<li>通过创建 2 毫米厚的水平部分，使用 0.009" 不锈钢、未涂层、单边剃须刀刀片，生成 6 个脑片。</li>
	<li>在 0.05% TTC 中在 37 °C 下孵化 30 分钟。</li>
	<li>将脑组织放在显微镜幻灯片上，对分辨率为 1600x1600 dpi 的这 6 个脑片进行光学扫描（例如，参见补充 1）。</li>
	<li>使用通道混频器功能（图像 + 调整 + 通道混频器） 添加带有照片编辑器的蓝色滤镜（例如 Adobe Photoshop CS2），并将图像保存为 JPEG 文件格式。 注:应用蓝色滤镜后，图像将出现灰度。</li>
	<li>在 ImageJ 1.37v29、30中打开保存的图像。 注:此计算机程序使用阈值函数来隔离和计算黑色或白色的像素（见图 1）。</li>
	<li>对于图像的 6 个大脑切片中的每一个，使用主菜单中的"多边形选择"工具，选择并保存每个半球（右侧受伤的 ipsilateral 和左侧未受伤的反面）作为单独的图像文件。</li>
	<li>通过选择 Image > 调整 + 阈值，使用 ImageJ 软件主菜单中的自动阈值功能设置确定 IZ 的截止值，并测量单个大脑集每个半球的像素数。 注:宏可用于 ImageJ 软件中的此步骤（请参阅代码 补充 2）。 切断是确定哪些像素转换为白色，哪些转换为黑色取决于灰色阴影的关键参数（见 补充 3 和 补充 4 作为示例）。然后，ImageJ 比较白色和黑色像素以确定 IZ。根据染色协议和扫描仪设置，我们使用了 0.220 的恒定截止值。</li>
	<li>使用益普西拉特尔和反三边脑半球（RICH）方法13、23（见补充5中的示例）对组织肿胀进行IZ校正测量。 <img alt="Equation 1" src="/files/ftp_upload/61309/61309equ01.jpg"> 注:梗死大小被评估为反半球的百分比。</li>
</ol>5. 脑水肿的确定31
注:使用图像J 1.37v测量 BE32，33。
<ol><li>测量是 24 小时后， Mcao 。要计算 BE，请使用来自左右半球体积（单位）的数据。</li>
	<li>执行分辨率为 1600x1600 dpi 的光学扫描（例如，请参阅补充 1）。</li>
	<li>选择大脑半球，并设置用 ImageJ 1.37v 确定 BE 的截止点，如上文在 4.17-4.19 节中所述。</li>
	<li>将 BE 区域作为未受影响的反向半球标准区域的百分比，由 RICH 方法使用以下方程计算（见补充 5中的示例）23、34。 <img alt="Equation 2" src="/files/ftp_upload/61309/61309equ02v2.jpg"> 注:BE 的程度被评估为反半球的百分比。</li>
</ol>6. BBB 中断的确定
<ol><li>测量 MCAO 后 24 小时的 BBB 中断。</li>
	<li>将左右半球分成六个切片，每个切片放入微中心管中。</li>
	<li>根据计算4mL中1克脑组织50%三氯乙酸的三氯乙酸，将三氯乙酸中的每一片脑组织同质化。</li>
	<li>离心机在10，000 x g 20分钟。</li>
	<li>用 96% 乙醇稀释超纳坦液体 1:3。</li>
	<li>利用光谱学软件进行发光光谱测量，安装板材，并使用以下参数进行样品读取:荧光强度激发波长为620nm（带宽10nm），发射波长为680nm（带宽10nm）23，35:模组顶部;弗莱什25号;手动 100;摇晃1秒1毫米 注:使用620nm（带宽10nm）和680nm（带宽10nm）的激发波长。23，35
</li>
</ol>

作者没有什么可透露的。

本议定书的主要目标是证明对缺血损伤的三个主要参数的一致测量:IZ、BE和BBB渗透性。先前在这一领域的研究表明，有可能在同一样本中一起执行其中一个或两个参数。除了这种由三部分组成的方法提供的成本降低外，它还提供了一个更理想的生物伦理模型，限制必须操作并随后实施安乐死的动物数量。与所有病理学技术一样，该方法受到无法动态观察缺血损伤的限制。
在图...

全世界发病率和死亡率最常见的原因之一是中风。在全球范围内，缺血性中风占所有中风病例的68%，而在美国缺血性中风占中风病例的87%。1，2。据估计，中风的经济负担达到340亿美元，在美国2和450亿欧元在欧盟3。动物中风模型是研究其病理生理学，开发新的评价方法，并提出新的治疗方案4。
缺血性中风发生在一个主要脑动脉的闭塞，通常是中脑动脉或其分支之一5。因此，缺血性中风的模型历来涉及中脑动脉闭塞（MCAO）6，7，8，9，10，11，12。继 MCAO 之后， 神经损伤最常通过测量梗塞区 （IZ） 来评估，使用 2，3，5 三苯甲酸酯氯化物 （TTC） 染色方法13，脑水肿 （BE） 使用 干燥或计算半球体积14，15，16，和血脑屏障（BBB）渗透性光谱技术使用埃文斯蓝色染色17，18，19。
传统的 MCAO 方法在三个大脑测量中每个都使用单独的大脑集。对于大量的样本量，这导致大量的安乐死动物，增加了伦理和财务考虑。减轻这些成本的替代方法将涉及在一组后 MCAO 啮齿动物大脑中测量所有三个参数。
先前曾尝试测量同一大脑样本中的参数组合。同时免疫荧光染色方法20 以及其他分子和生化分析21 已描述后TTC染色在同一大脑样本。我们以前计算过脑半球的体积来评估脑水肿，并进行了TTC染色，以计算同一大脑集15的梗塞区。
在本协议中，我们提出了一种经过改进的 MCAO 技术，通过确定同一组啮齿动物大脑中的 IZ、BE 和 BBB 渗透性来测量缺血性脑损伤。IZ通过TTC染色测量，BE通过计算半球体积来测量，BBB渗透性通过光谱方法19获得。在此协议中，我们使用了经过修改的 MCAO 模型，基于将单滤导管直接插入和固定到内部胡萝卜动脉 （ICA） 和进一步阻断流向中脑动脉 （MCA）22的血液。与传统的MCAO方法16、22相比，这种修改后的方法表明死亡率和发病率有所下降。
这种新方法为测量 MCAO 之后的神经损伤提供了财务健全和道德模型。这种对缺血性脑损伤主要参数的评估将有助于全面研究其病理生理学。

<table><tbody><tr><td>2 mL Syringe</td><td>Braun</td><td>4606027V</td><td></td></tr><tr><td>2% chlorhexidine in 70% alcohol solution</td><td>Sigma-Aldrich</td><td>500 cc</td><td>Provides general antisepsis of the skin in the operatory field</td></tr><tr><td>27 G Needle with Syringe</td><td>Braun</td><td>305620</td><td></td></tr><tr><td>3-0 Silk sutures</td><td>Henry Schein</td><td>1007842</td><td></td></tr><tr><td>4-0 Nylon suture</td><td>4-00</td><td></td><td></td></tr><tr><td>Brain &amp;amp; Tissue Matrices</td><td>Sigma-Aldrich</td><td>15013</td><td></td></tr><tr><td>Cannula Venflon 22 G</td><td>KD-FIX</td><td>183603985447</td><td></td></tr><tr><td>Centrifuge Sigma 2-16P</td><td>Sigma-Aldrich</td><td>Sigma 2-16P</td><td></td></tr><tr><td>Compact Analytical Balances</td><td>Sigma-Aldrich</td><td>HR-AZ/HR-A</td><td></td></tr><tr><td>Digital weighing scale</td><td>Sigma-Aldrich</td><td>Rs 4,000</td><td></td></tr><tr><td>Dissecting scissors</td><td>Sigma-Aldrich</td><td>Z265969</td><td></td></tr><tr><td>Eppendorf pipette</td><td>Sigma-Aldrich</td><td>Z683884</td><td></td></tr><tr><td>Eppendorf tube</td><td>Sigma-Aldrich</td><td>EP0030119460</td><td></td></tr><tr><td>Fluorescence detector</td><td>Tecan, M&amp;auml;nnedorf Switzerland</td><td>Model: Infinite 200 PRO multimode reader</td><td>Optional.</td></tr><tr><td>Fluorescence detector</td><td>Molecular Devices LLC</td><td>VWR cat. # 10822 512 SpectraMax Paradigm Multi Mode Microplate Reader Base Instrument</td><td>Optional.</td></tr><tr><td>Gauze sponges</td><td>Fisher</td><td>22-362-178</td><td></td></tr><tr><td>Heater with thermometer</td><td>Heatingpad-1</td><td>Model: HEATINGPAD-1/2</td><td></td></tr><tr><td>Hemostatic microclips</td><td>Sigma-Aldrich</td><td></td><td></td></tr><tr><td>Horizon-XL</td><td>Mennen Medical Ltd</td><td></td><td></td></tr><tr><td>Infusion cuff</td><td>ABN</td><td>IC-500</td><td></td></tr><tr><td>Micro forceps</td><td>Sigma-Aldrich</td><td></td><td></td></tr><tr><td>Micro scissors</td><td>Sigma-Aldrich</td><td></td><td></td></tr><tr><td>Multiset</td><td>Teva Medical</td><td>998702</td><td></td></tr><tr><td>Olympus BX 40 microscope</td><td>Olympus</td><td></td><td></td></tr><tr><td>Operating forceps</td><td>Sigma-Aldrich</td><td></td><td></td></tr><tr><td>Operating scissors</td><td>Sigma-Aldrich</td><td></td><td></td></tr><tr><td>Optical scanner</td><td>Canon</td><td>Cano Scan 4200F</td><td>Resolution 3200 x 6400 dpi</td></tr><tr><td>Petri dishes</td><td>Sigma-Aldrich</td><td>P5606</td><td></td></tr><tr><td>Purina Chow</td><td>Purina</td><td>5001</td><td>Rodent laboratory chow given to rats, mice and hamster is a life-cycle nutrition that has been used in biomedical research for over 5 decades. Provided to rats ad libitum in this experiment.</td></tr><tr><td>Rat cages</td><td>Techniplast</td><td>2000P</td><td>Conventional housing for rodents. Cages were used for housing rats throughout the experiment</td></tr><tr><td>Scalpel blades #11</td><td>Sigma-Aldrich</td><td>S2771</td><td></td></tr><tr><td>&lt;strong&gt;Software&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Adobe Photoshop CS2 for Windows</td><td>Adobe</td><td></td><td></td></tr><tr><td>ImageJ 1.37v</td><td>NIH</td><td></td><td>The source code is freely available. The author, Wayne Rasband (wayne@codon.nih.gov), is at the Research Services Branch, National Institute of Mental Health, Bethesda, Maryland, USA</td></tr><tr><td>SPSS Statistics 22</td><td>IBM</td><td></td><td></td></tr><tr><td>Office 365 ProPlus</td><td>Microsoft</td><td>-</td><td>Microsoft Office Excel</td></tr><tr><td>Windows 10</td><td>Microsoft</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>2,3,5-Triphenyltetrazolium chloride</td><td>Sigma-Aldrich</td><td>298-96-4</td><td></td></tr><tr><td>50% trichloroacetic acid</td><td>Sigma-Aldrich</td><td>76-03-9</td><td></td></tr><tr><td>Ethanol 96 %</td><td>Romical</td><td></td><td>Flammable liquid</td></tr><tr><td>Evans blue 2%</td><td>Sigma-Aldrich</td><td>314-13-6</td><td></td></tr><tr><td>Isoflurane, USP 100%</td><td>Piramamal Critical Care, Inc</td><td>NDC 66794-017</td><td></td></tr></tbody></table>

measuring post-stroke cerebral edema, infarct zone and blood-brain barrier breakdown in a single set of rodent brain samples

全世界发病率和死亡率最常见的原因之一是缺血性中风。从历史上看，用于刺激缺血性中风的动物模型涉及中脑动脉闭塞（MCAO）。梗塞区、脑水肿和血脑屏障 （BBB） 故障被测量为反映 MCAO 后脑损伤程度的参数。这种方法的一个重大限制是，这些测量通常在不同的大鼠大脑样本中获得，由于大量大鼠需要安乐死以达到适当的样本大小，因此会导致伦理和经济负担。在这里，我们提出了一种方法，通过测量同一组大鼠大脑中的梗塞区、脑水肿和BBB渗透率，准确评估MCAO之后的脑损伤。这种新技术为评估中风的病理生理学提供了更有效的方法。

梗塞区测量
一项独立样本T测试表明，与16只假手术大鼠相比，19只接受永久性MCAO的老鼠的大脑梗塞体积显著增加（MCAO=7.49%，±3.57对。 香=0.31%±1.9，t（28.49）=7.56，p<0.01（见图2A）。 数据表示为反半球± SD 的平均百分比。
脑水肿测量
一项独立样本T测试表明，与16只假手术大...

Watch this Scientific Journal Video about Measuring Post-Stroke Cerebral Edema, Infarct Zone and Blood-Brain Barrier Breakdown in a Single Set of Rodent Brain Samples at JoVE.com

Measuring Post-Stroke Cerebral Edema, Infarct Zone and Blood-Brain Barrier Breakdown in a Single Set of Rodent Brain Samples

此协议描述了在同一组啮齿动物脑样本上测量缺血性脑损伤三个最重要的参数的新技术。仅使用一个大脑样本在道德和经济成本方面非常有利。

测量一组啮齿动物脑样本中的中风后脑水肿、梗塞区和血脑屏障破裂

One of the most common causes of morbidity and mortality worldwide is ischemic stroke. Historically, an animal model used to stimulate ischemic stroke ...

measuring-post-stroke-cerebral-edema-infarct-zone-blood-brain-barrier

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

7.8K 次观看.  Ben-Gurion University of the Negev. 此协议描述了在同一组啮齿动物脑样本上测量缺血性脑损伤三个最重要的参数的新技术。仅使用一个大脑样本在道德和经济成本方面非常有利。

视频: Measuring Post-Stroke Cerebral Edema, Infarct Zone and Blood-Brain Barrier Breakdown in a Single Set of Rodent Brain Samples

Focal Cerebral Ischemia Model by Endovascular Suture Occlusion of the Middle Cerebral Artery in the Rat

Stroke is the leading cause of disability and the third leading cause of death in adults worldwide1. In human stroke, there exists a highly variable clinical state; in the development of animal models of focal ischemia, however, achieving reproducibility of experimentally induced infarct volume is essential. The rat is a widely used animal model for stroke due to its relatively low animal husbandry costs and to the similarity of its cranial circulation to that of humans2,3. In humans, the middle cerebral artery (MCA) is most commonly affected in stroke syndromes and multiple methods of MCA occlusion (MCAO) have been described to mimic this clinical syndrome in animal models. Because recanalization commonly occurs following an acute stroke in the human, reperfusion after a period of occlusion has been included in many of these models. In this video, we demonstrate the transient endovascular suture MCAO model in the spontaneously hypertensive rat (SHR). A filament with a silicon tip coating is placed intraluminally at the MCA origin for 60 minutes, followed by reperfusion. Note that the optimal occlusion period may vary in other rat strains, such as Wistar or Sprague-Dawley. Several behavioral indicators of stroke in the rat are shown. Focal ischemia is confirmed using T2-weighted magnetic resonance images and by staining brain sections with 2,3,5-triphenyltetrazolium chloride (TTC) 24 hours after MCAO.

Surgical induction of ischemic brain damage in the rat is a widely used model for stroke research. Here we demonstrate the induction of focal cerebral ischemia by occlusion of the middle cerebral artery. Visualization of the resulting infarct by histological staining and magnetic resonance imaging is also shown.

焦距脑缺血模型大鼠大脑中动脉的血管内缝合闭塞

Stroke is the leading cause of disability and the third leading cause of death in adults worldwide1. In human stroke, there exists a highly variable ...

科研

神经科学

2-Vessel Occlusion/Hypotension: A Rat Model of Global Brain Ischemia

Cardiac arrest followed by resuscitation often results in dramatic brain damage caused by ischemia and subsequent reperfusion of the brain. Global brain ischemia produces damage to specific brain regions shown to be highly sensitive to ischemia 1. Hippocampal neurons have higher sensitivity to ischemic insults compared to other cell populations, and specifically, the CA1 region of the hippocampus is particularly vulnerable to ischemia/reperfusion 2.
The design of therapeutic interventions, or study of mechanisms involved in cerebral damage, requires a model that produces damage similar to the clinical condition and in a reproducible manner. Bilateral carotid vessel occlusion with hypotension (2VOH) is a model that produces reversible forebrain ischemia, emulating the cerebral events that can occur during cardiac arrest and resuscitation. We describe a model modified from Smith et al. (1984) 2, as first presented in its current form in Sanderson, et al. (2008) 3, which produces reproducible injury to selectively vulnerable brain regions 3-6. The reliability of this model is dictated by precise control of systemic blood pressure during applied hypotension, the duration of ischemia, close temperature control, a specific anesthesia regimen, and diligent post-operative care. An 8-minute ischemic insult produces cell death of CA1 hippocampal neurons that progresses over the course of 6 to 24 hr of reperfusion, while less vulnerable brain regions are spared. This progressive cell death is easily quantified after 7-14 days of reperfusion, as a near complete loss of CA1 neurons is evident at this time.
In addition to this brain injury model, we present a method for CA1 damage quantification using a simple, yet thorough, methodology. Importantly, quantification can be accomplished using a simple camera-mounted microscope, and a free ImageJ (NIH) software plugin, obviating the need for cost-prohibitive stereology software programs and a motorized microscopic stage for damage assessment.

Bilateral carotid occlusion coupled with systemic hypotension produces global brain ischemia in the rat, resulting in damage to the hippocampus with reproducible severity. Animal subjects are impaired with predictable patterns of brain damage, they recover expediently, and mortality rates are comparatively low.

2血管闭塞/低血压：全脑缺血模型大鼠

Cardiac arrest followed by resuscitation often results in dramatic brain damage caused by ischemia and subsequent reperfusion of the brain. Global brain ...

医学

Optimized System for Cerebral Perfusion Monitoring in the Rat Stroke Model of Intraluminal Middle Cerebral Artery Occlusion

The translational potential of pre-clinical stroke research depends on the accuracy of experimental modeling. Cerebral perfusion monitoring in animal models of acute ischemic stroke allows to confirm successful arterial occlusion and exclude subarachnoid hemorrhage. Cerebral perfusion monitoring can also be used to study intracranial collateral circulation, which is emerging as a powerful determinant of stroke outcome and a possible therapeutic target. Despite a recognized role of Laser Doppler perfusion monitoring as part of the current guidelines for experimental cerebral ischemia, a number of technical difficulties exist that limit its widespread use. One of the major issues is obtaining a secure and prolonged attachment of a deep-penetration Laser Doppler probe to the animal skull. In this video, we show our optimized system for cerebral perfusion monitoring during transient middle cerebral artery occlusion by intraluminal filament in the rat. We developed in-house a simple method to obtain a custom made holder for twin-fibre (deep-penetration) Laser Doppler probes, which allow multi-site monitoring if needed. A continuous and prolonged monitoring of cerebral perfusion could easily be obtained over the intact skull.

Cerebral perfusion monitoring has been demonstrated to improve accuracy in ischemic stroke models. Technical difficulties often limit the use of this essential tool for cerebrovascular research. In this video, an optimized system is shown to obtain a single or multi-site hemodynamic monitoring during intraluminal middle cerebral artery occlusion in rats.

优化系统的脑灌注监测在中风模型大鼠的大脑中动脉闭塞腔内

The translational potential of pre-clinical stroke research depends on the accuracy of experimental modeling. Cerebral perfusion monitoring in animal ...

A Middle Cerebral Artery Occlusion Technique for Inducing Post-stroke Depression in Rats

Post-stroke depression (PSD) is the most recurrent of all psychiatric complications resulting from an ischemic stroke. A greater majority (about 60%) of all ischemic stroke patients suffer from PSD, a disorder considered to be an ischemic stroke-related precursor for increased death and degradation in health. The pathophysiology of PSD is still obscure. To study the mechanism of development and occurrence of PSD further, and to find out a therapy, we attempted to develop a new protocol that requires occluding the middle cerebral artery (MCA) via the internal carotid artery (ICA) in rats. This protocol describes a model of PSD induced in rats through the middle cerebral artery occlusion (MCAO). Also used in the experiment are the Porsolt forced swim test and the sucrose preference test to confirm and evaluate the depressive mood of the rats under investigation. Rather than inserting the catheter through the external carotid artery (ECA), as stipulated for the original procedure, this MCAO technique has the monofilament passing directly through the ICA. This MCAO technique was developed a few years ago and leads to a reduction in mortality and variability. It is generally accepted that the criteria used are preferred in the selection of biological models. The data obtained with this protocol show that this model of MCAO could be a way of inducing PSD in rats and could potentially lead to the understanding of the pathophysiology and the future development of new drugs and other neuroprotective agents.

Here we present a protocol to induce post-stroke depression in rats by occluding the middle cerebral artery via the internal carotid artery. We use the Porsolt forced swim test and the sucrose preference test to confirm and evaluate induced depressive moods.

一种中脑动脉闭塞技术在大鼠脑卒中后抑郁中的诱导

Post-stroke depression (PSD) is the most recurrent of all psychiatric complications resulting from an ischemic stroke. A greater majority (about 60%) of ...

行为学

Stereological and Flow Cytometry Characterization of Leukocyte Subpopulations in Models of Transient or Permanent Cerebral Ischemia

Microglia activation, as well as extravasation of haematogenous macrophages and neutrophils, is believed to play a pivotal role in brain injury after stroke. These myeloid cell subpopulations can display different phenotypes and functions and need to be distinguished and characterized to study their regulation and contribution to tissue damage. This protocol provides two different methodologies for brain immune cell characterization: a precise stereological approach and a flow cytometric analysis. The stereological approach is based on the optical fractionator method, which calculates the total number of cells in an area of interest (infarcted brain) estimated by a systematic random sampling. The second characterization approach provides a simple way to isolate brain leukocyte suspensions and to characterize them by flow cytometry, allowing for the characterization of microglia, infiltrated monocytes and neutrophils of the ischemic tissue. In addition, it also details a cerebral ischemia model in mice that exclusively affects brain cortex, generating highly reproducible infarcts with a low rate of mortality, and the procedure for histological brain processing to characterize infarct volume by the Cavalieri method.

Brain myeloid cells characterization following stroke can be performed by stereology using the optical fractionator method, or by a flow cytometric analysis of brain leukocytes suspensions. Both are useful techniques to perform an accurate phenotypical distinction of the main myeloid cell subsets found in the ischemic brain.

白细胞亚群在暂时或永久的脑缺血模型体视学和流式细胞仪鉴定

Microglia activation, as well as extravasation of haematogenous macrophages and neutrophils, is believed to play a pivotal role in brain injury after ...

Quantification of Neurovascular Protection Following Repetitive Hypoxic Preconditioning and Transient Middle Cerebral Artery Occlusion in Mice

Experimental animal models of stroke are invaluable tools for understanding stroke pathology and developing more effective treatment strategies. A 2 week protocol for repetitive hypoxic preconditioning (RHP) induces long-term protection against central nervous system (CNS) injury in a mouse model of focal ischemic stroke. RHP consists of 9 stochastic exposures to hypoxia that vary in both duration (2 or 4 hr) and intensity (8% and 11% O2). RHP reduces infarct volumes, blood-brain barrier (BBB) disruption, and the post-stroke inflammatory response for weeks following the last exposure to hypoxia, suggesting a long-term induction of an endogenous CNS-protective phenotype. The methodology for the dual quantification of infarct volume and BBB disruption is effective in assessing neurovascular protection in mice with RHP or other putative neuroprotectants. Adult male Swiss Webster mice were preconditioned by RHP or duration-equivalent exposures to 21% O2 (i.e. room air). A 60 min transient middle cerebral artery occlusion (tMCAo) was induced 2 weeks following the last hypoxic exposure. Both the occlusion and reperfusion were confirmed by transcranial laser Doppler flowmetry. Twenty-two hr after reperfusion, Evans Blue (EB) was intravenously administered through a tail vein injection. 2 hr later, animals were sacrificed by isoflurane overdose and brain sections were stained with 2,3,5- triphenyltetrazolium chloride (TTC). Infarcts volumes were then quantified. Next, EB was extracted from the tissue over 48 hr to determine BBB disruption after tMCAo. In summary, RHP is a simple protocol that can be replicated, with minimal cost, to induce long-term endogenous neurovascular protection from stroke injury in mice, with the translational potential for other CNS-based and systemic pro-inflammatory disease states.

This protocol describes repetitive hypoxic preconditioning, or brief exposures to systemic hypoxia that reduce infarct volumes and blood-brain barrier disruption following transient middle cerebral artery occlusion in mice. It also details dual quantification of infarct volume and blood-brain barrier disruption after stroke to assess the efficacy of neurovascular protection.

神经血管保护定量继在小鼠重复缺氧预处理和瞬态大脑中动脉闭塞

Experimental animal models of stroke are invaluable tools for understanding stroke pathology and developing more effective treatment strategies. A 2 week ...

An In Vivo Assessment of Blood-Brain Barrier Disruption in a Rat Model of Ischemic Stroke

Ischemic stroke leads to vasogenic cerebral edema and subsequent primary brain injury, which is mediated through destruction of the blood-brain barrier (BBB). Rats with induced ischemic stroke were established and used as in vivo models to investigate the functional integrity of the BBB. Spectrophotometric detection of Evans blue (EB) in the brain samples with ischemic injury could provide reliable justification for the research and development of novel therapeutic modalities. This method generates reproducible results, and is applicable in any laboratory without a need for special equipment. Here, we present a visualized and technical guideline on the detection of the extravasation of EB following induction of ischemic stroke in rats.

An In Vivo Assessment of Blood-Brain Barrier Disruption in a Rat Model of Ischemic Stroke

The overall goal of this procedure is to provide a highly reproducible technique for in vivo assessment of the blood-brain barrier disruption in rat models of ischemic stroke.

一种体内评价大鼠缺血性中风模型中血脑屏障的破坏

Ischemic stroke leads to vasogenic cerebral edema and subsequent primary brain injury, which is mediated through destruction of the blood-brain barrier ...

Laser-Induced Brain Injury in the Motor Cortex of Rats

A common technique for inducing stroke in experimental rodent models involves the transient (often denoted as MCAO-t) or permanent (designated as MCAO-p) occlusion of the middle cerebral artery (MCA) using a catheter. This generally accepted technique, however, has some limitations, thereby limiting its extensive use. Stroke induction by this method is often characterized by high variability in the localization and size of the ischemic area, periodical occurrences of hemorrhage, and high death rates. Also, the successful completion of any of the transient or permanent procedures requires expertise and often lasts for about 30 minutes. In this protocol, a laser irradiation technique is presented that can serve as an alternative method for inducing and studying brain injury in rodent models.
When compared to rats in the control and MCAO groups, the brain injury by laser induction showed reduced variability in body temperature, infarct volume, brain edema, intracranial hemorrhage, and mortality. Furthermore, the use of a laser-induced injury caused damage to the brain tissues only in the motor cortex unlike in the MCAO experiments where destruction of both the motor cortex and striatal tissues is observed.
Findings from this investigation suggest that laser irradiation could serve as an alternative and effective technique for inducing brain injury in the motor cortex. The method also shortens the time for completing the procedure and does not require expert handlers.

The protocol presented here shows a technique to create a rodent model of brain injury. The method described here uses laser irradiation and targets motor cortex.

大鼠运动皮层中的激光诱发脑损伤

A common technique for inducing stroke in experimental rodent models involves the transient (often denoted as MCAO-t) or permanent (designated as MCAO-p) ...

A Fibrin-Enriched and tPA-Sensitive Photothrombotic Stroke Model

An ideal thromboembolic stroke model requires certain properties, including relatively simple surgical procedures with low mortality, a consistent infarction size and location, precipitation of platelet:fibrin intermixed blood clots similar to those in patients, and an adequate sensitivity to fibrinolytic treatment. The rose bengal (RB) dye-based photothrombotic stroke model meets the first two requirements but is highly refractory to tPA-mediated lytic treatment, presumably due to its platelet-rich, but fibrin-poor clot composition. We reason that combination of RB dye (50 mg/kg) and a sub-thrombotic dose of thrombin (80 U/kg) for photoactivation aimed at the proximal branch of middle cerebral artery (MCA) may produce fibrin-enriched and tPA-sensitive clots. Indeed, the thrombin and RB (T+RB)-combined photothrombosis model triggered mixed platelet:fibrin blood clots, as shown by immunostaining and immunoblots, and maintained consistent infarct sizes and locations plus low mortality. Moreover, intravenous injection of tPA (Alteplase, 10 mg/kg) within 2 h post-photoactivation significantly decreased the infarct size in T+RB photothrombosis. Thus, the thrombin-enhanced photothrombotic stroke model may be a useful experimental model to test novel thrombolytic therapies.

Traditional photothrombotic stroke (PTS) models mainly induce dense platelet aggregates of a high resistance to tissue plasminogen activator (tPA)-lytic treatment. Here a modified murine PTS model is introduced by co-injecting thrombin and photosensitive dye for photoactivation. The thrombin-enhanced PTS model produces mixed platelet:fibrin clots and is highly sensitive to tPA-thrombolysis.

纤维蛋白富集和 tPA 敏感的光血栓形成卒中模型

An ideal thromboembolic stroke model requires certain properties, including relatively simple surgical procedures with low mortality, a consistent ...

Early Pathological and Magnetic Resonance Detection of Cerebral Injury Using a Rat Model of Neonatal Hypoxic Ischemic Encephalopathy

Perinatal hypoxic-ischemic encephalopathy (HIE) is an acute disease that may afflict newborns, resulting in variable long- and short-term neurodevelopmental outcomes. Early diagnosis is critical to identifying infants who may benefit from intervention; however, early diagnosis relies heavily on clinical criteria. No molecular or radiological tests have shown promise in detecting early cerebral injury. Studies have shown that magnetic resonance imaging (MRI) can show changes in both blood flow/ischemia and metabolic disruption. However, they have all been used to evaluate the secondary phase of the disease (&gt;12 h) after the onset of the injury. Early diagnosis is critical to rapidly starting therapeutic hypothermia in eligible infants, which is currently recommended to be initiated within 6 h of birth. The rat model of hypoxic-ischemic injury was developed in 1981 and has been validated and used extensively to study changes in brain perfusion, cerebral injury markers, and morphology. However, it has primarily been used as a "late model", evaluating injury several days after the initial ischemic insult. The model has been known to have poor sensitivity in evaluating reliable and reproducible early cerebral changes. The objective of this study was to develop a reliable model to study early gross morphological and radiological markers of HIE using pathological staining and cerebral magnetic resonance imaging/magnetic resonance spectroscopy.

The present protocol describes a rodent model of newborn hypoxic-ischemic injury for identifying early changes in cerebral tissue by gross morphology and magnetic resonance imaging. This has benefits over existing models, which can be used to study late injury but do not allow the evaluation of reproducible early changes.